In vitro analysis of volatile organic compounds in search of potential biomarkers of lung cancer

Lung cancer is a leading cause of death from cancer worldwide. An early diagnosis and appropriate treatment are crucial in reducing mortality among people suffering from the disease. Therefore, one of the main focuses of lung cancer studies is on advances in its early detection. One of the most promising is the analysis of volatile organic compounds (VOCs). VOCs are a diverse group of carbon-based chemicals that are present in exhaled breath and biofluids, and may be collected from the headspace of these matrices. Different patterns of VOCs have been correlated with various diseases, cancer among them. Studies have shown that various cancer cells in vitro produce or consume specific VOCs that can serve as potential biomarkers to differentiate them from non-cancer cells. The present study aimed at the detection, identification and semi-quantification of VOCs released or consumed by the adenocarcinoma human alveolar A549 cell line. For this purpose, gas chromatography with mass spectrometric detection was combined with two pre-concentration techniques: monolithic material sorptive extraction (MMSE) or extraction of thermal desorption (TD) sorbent tubes with an Easy-VOCTM pump as a sample loading tool. MMSE is a new technique for the extraction of VOCs from various samples and it is used for the first time for the analysis of VOCs from cell culture medium. TD-GC-MS is a popular technique for VOCs analysis and it has been used for the first time here with Bio-VOCTM pump in the studies of VOCs in vitro. The project also aimed at the comparison of the A549 VOC level trends to the trends of the normal human lung fibroblasts NHLF (MMSE experiment) and normal human bronchial BEAS-2B cells (TD experiment). In addition, the VOC patterns between the growing and confluent cells of the same cell line were compared for the first time. In the MMSE experiment, seven VOCs were produced and 14 VOCs metabolised exclusively by the cancer cells. Among the released compounds were methylated hydrocarbons (2,4-dimethyl-1-heptene; 4-methylundecane; 2,3,6,7-tetramethyloctane 2,3,5-trimethylhexane and 2,3,5-trimethyldecane) and alcohols (cyclohexanol and 3- heptanol). The metabolised analytes were alcohols (4-decanol; 6-dodecanol; 2- ethylhexanol; 1-octanol), aldehydes (dodecanal; tetradecanal), ketones (acetophenone; cyclohexanone; 2-tetradecanone), phenols (phenol and 2-nitrophenol); an ether (2-methoxydiphenylmethane), an ester (pentanoic acid, 2,4-dimethyl-3-oxo-, methyl ester) and a hydrocarbon (tetradecane). 2,4-Dimethylheptane; 2,6-di-tert-butyl-1,4- benzoquinone; 1-phenylethanol and 2-pentadecanone were released by both A549 and NHLF cell lines. The cancer cells were observed, however, to emit the VOCs at a higher level than the fibroblasts. Benzaldehyde; 2-ethylhexanol; hexanal and 1-nonanol were found to be consumed by both the cancer and NHLF cells, however, at a greater rate by the former. In the TD experiment, 2,3,5-trimethylhexane and tert-butanol were produced exclusively by the A549 cells, while ethyl acetate solely by the BEAS-2B cells. Acetophenone, benzaldehyde and 2-methylbutanal were metabolised and acetone was produced at higher levels by the cancer cells than by the BEAS-2B cells. The possible use of the analysed VOCs as potential biomarkers of lung cancer is discussed, along with the suggestion and discussion of the possible metabolic pathways leading to the uptake and release of these VOCs by the analysed cells. Also the discussion of poor correlation between different in vitro studies, as well as between in vivo and in vitro studies of VOCs as potential biomarkers of cancer, is undertaken